Type of paper: Report

Topic: Protein, Gel, DNA, Education, Medicine, The Matrix, Serum, Staining

Pages: 7

Words: 1925

Published: 2020/11/01

New WowEssays Premium Database!

Find the biggest directory of over
1 million paper examples!

Introduction

Polyacrylamide gel electrophoresis is a fundamental technique applicable in most biological investigations ranging from biochemistry to forensic. The mechanism is used for the separation of macromolecules especially proteins together with nucleic acids with respect to the electrophoretic mobility of the molecules being separated.
Electrophoresis is a relatively simple method to study the properties of proteins. It is based on the principle that charged molecules will migrate through a matrix when an electric field is applied. The matrix used is polyacrylamide. NB Polyacrylamide is highly toxic, carcinogenic and teratogenic. The chemical agents used to form the polyacrylamide matrix are acrylamide monomer and N, N methylene bisacrylamide. The size of the pores created in the matrix depends on the amount of acrylamide per unit volume and the percentage of the bisacrylamide. The normal range of polyacrylamide gels is 3-30%. Protein electrophoresis using polyacrylamide is sometimes called PAGE: polyacrylamide gel electrophoresis.
There are two fundamentally different PAGE systems. These are dissociative (denaturing) and non-dissociative (non-denaturing). In non-dissociative PAGE, the proteins are maintained in their native conformations. Dissociative PAGE is designed to denature the proteins into their constituent polypeptides. This is usually done using a detergent called sodium dodecyl sulfate and hence the technique is called SDS-PAGE. This is the most common method for separating proteins based exclusively on size.

Objective

This exercise aimed at understanding the principles of electrophoresis, the use of protein standards for calculating protein mass and the difference between denaturing and non-denaturing gel electrophoresis.

Method

Since the gel was not prepared in this lab because, a ready and prepared gel was used. The resolving gel was cast 4-12% BT (NU-PAGE). A volume of the sample that contains 5ug of protein was pipetted into an Eppendorf tube (the concentration of the sample was calculated in lab 1). 2.5ul of buffer and 2.5ul of water was added to make the total sample 10ul. The sample mixture was then heated for two minutes before loading it onto the gel along with protein standards provided. The gel was resolved at 150mv for 1 hour. The gel was dismantled and stained in Coomassie blue solution (instant blue) for 5 minutes at the end of the time. Then the gel was destained and dried.

Discussion

Describe the principle of electrophoretic separation of proteins (5marks)
The principle used in electrophoretic separation is the migration aspect of the charged molecules within the electric field. The consideration of difference in size of the molecules, charge or conformation features that influence the mobility of the molecules in the gel is critical. Whenever the molecules which are charged are put in an electric field, depending on the charge they possess they move towards either the negative or the positive pole. This is contrary to the proteins that can have a net of the positive or negative charge, the nucleic acids have a phosphate backbone which convey a negative charge on them and they migrate towards the positive pole as a result. The molecules are electrophosed within a matrix. The molecules migrate to different distances in the gel depending on their mobility. Therefore, the molecules are separated according to their mobility in the matrix.
what is the role of the following in SDS in SDS-PAGE:

SDS

sodium dodecyl sulfate functions as a detergent that denatures the protein by dissolving the hydrophobic molecules before it is loaded to the sodium dodecyl sulfate polyacrylamide gel electrophoresis. The denatured protein migrates through the polyacrylamide – the matrix.

B-mercaptoethanol

B-mercaptoethanol is incorporated in the buffer so as to cleave the disulfide links within or else amid the molecules, letting molecules to assume an extended monomeric conformation.

TEMED and ammonium persulphate (5 marks)

TEMED is a stabilizer of free radicals and has the role of enhancing polymerization. The ammonium persulfate (APS) assists in inducing the polymerization of acrylamide and bisacrylamide monomers. The ammonium persulfate decomposes rapidly to form free radicals.
Research the relationship between molecular weight and distance travelled on the gel. Using the model data supplied, calculate the weight of the unknown protein (20)
The comparative rate of mobility relies upon the charge, molar weight, as well as the shape of the protein analyzed. The proteins with a higher negative charge are attracted more readily and faster toward the anode, and thus move quickly. The gel medium enforces a separating influence upon the migration of protein molecules; the greater the protein the sluggish its migration, and small globular proteins move more rapidly than large elongated proteins.
4) Describe the gel matrix and its effects on the migration of proteins.
The gel matrix influences the rate of movement of proteins. The smaller gel takes shorter time to migrate and little reagents rather than the larger gels. The movement of proteins in the matrix is easier. The larger gel matrix takes longer time to migrate but ensure a higher resolution.
The recipe given is for a 12% gel. List the reagents and their volumes for a 10% gel. (5)
5ug of protein = 4.2ug
2.5ul of buffer = 2.1ul
2.5ul of water to make the total sample 10ul = 2.1ul make the total sample 8.3ul
5) Describe how you would modify the procedure if you wanted to study native protein conformation (3).
The native proteins are inactive according to their conformation. It is appropriate to unfold their chains into simpler and linear forms that are detectable. The unfolding of the complex form of the native protein can be done by denaturation. For this reason, the step for denaturation should be included in the procedure.
6) Describe the significance of the pH change between the two gels (2)
The pH change is critical in changing the counter- ions in the course of the runs so as to enhance the resolution.
7) We used Coomassie blue staining of proteins. Describe two other general protein stains. Under what conditions would they be used? (4 marks)

Silver Staining

Involves silver in staining histologic parts. The staining is essential especially in showing proteins such as collagen III, as well as the DNA. The staining is used in the situation where there is a need to identify substances within the cell and those that are outside.

Haematoxylin and eosin (H&E) staining

The two main constituents have different responsibilities whereby haematoxylin stains the nucleus RNA portions into a blue colour whilst the eosin stains other parts such as the cytoplasm, the substances suspended in the extracellular matrix and the connective tissue into pink or a deeper color (red). The technique of H and E staining is used in thin parts of the tissue.
8) Research and describe
a) 2D electrophoresis.
Complex components of one sample can be determined most absolutely by the two-dimensional electrophoresis (also known as 2D-PAGE). The initial dimension is responsible for separating proteins with respect to their natural isoelectric point (pI) and in this process, a type of electrophoresis used isoelectric focusing (IEF). The second dimension of the electrophoresis separates the sample components by mass through the use of ordinary SDS-PAGE. 2D PAGE avails the greatest resolution that is necessary for protein analysis.
The 2D electrophoresis is a critical technique in proteomic study and it is applicable to the situations where the resolution of numerous proteins on one gel is needed at times.
b) Western blotting. In what situations might you use these? (12 marks)
Western blotting is a method employed in the laboratory to recognize particular protein molecules among a complex mixture of proteins. The first phase of the process involves mixing the protein with sodium dodecyl sulfate to prepare it by unfolding it into linear simpler chains and coats together with negative charges. Molecules of the protein are then separated with respect to their sizes through gel electrophoresis. The protein molecules are then transferred from the gel to the blotting membrane due to the separation. The membrane carries all the protein bands initially present in the matrix after the migration is complete. The membrane then undergoes the blocking process to prevent unauthorized reactions from taking place. Then the incubation of the membrane takes place with a primary antibody which, then binds specifically to the targeted protein. The excess and unbound primary antibody is washed away before the sample is incubated with a secondary antibody to recognize and bind to the primary antibody. The secondary antibody is connected to a reporter protein that reacts to produce a different color that makes it visible hence can be identified.
Western blotting is used in the situation where there is need to identify a single protein molecules in a sample containing multiple species of proteins.
9) Describe two clinical uses of protein electrophoresis. (10 marks)

Serum Protein electrophoresis test

This is a clinical process that involves measuring of particular proteins present in the blood to assist in detecting some diseases. Proteins are the building portions of the body and are made up of amino acids which, are charged. The proteins consist of both positive and negative charges and move on the matrix when placed in an electric field. The serum protein electrophoresis applies the principle of electrical field to separate the constituent proteins contained in the blood serum into variant groups considering their characteristics such as shape and size as well as the charge. The blood serum mainly consists of albumin and globulin as the main constituents of protein origin. Both components are responsible for the transportation of substances via the bloodstream. The serum protein electrophoresis divides the two main proteins into the serum into five smaller constituents namely albumin, Alpha-1 globulin, Alpha-2 globulin, Beta globulin and Gamma globulin. Since the five proteins move at different rates in the electric field, they form a definite pattern that assist in the recognition of some diseases. For this reason, the further separation allows for more detailed analysis.

Clinical diagnosis of urine related infections

Alternative laboratory tests may result into abnormal results and as a result, protein electrophoresis is recommended as a follow-up test to ascertain the abnormal findings of the previous tests concerning the symptoms of a disease. The diagnosis may be followed by regular intervals of scrutinizing the trends of the disease together with the efficiency of treatment. Consider abnormalities in the urine protein levels, the test might be recommended to determine the cause of the higher concentrations in the urine. The technique can be applied in determining the source of protein and that if it is emanating from the blood plasma then this implies that the functioning of the kidney is altered. In case there is a proof that the excess protein is emanating from then cancer might be predicted in the cells. Other tests such as immunofixation electrophoresis can be applied for confirmation in the case of multiple monoclonal immunoglobulins.

Conclusion

The experiment achieved all its predetermined objectives successfully. The principles of electrophoresis have been explained precisely, and relevant questions addressed. Protein standards have been evaluated and understanding created through the explanation and mathematical evaluations. The different forms of gel electrophoreses have been elucidated through valid reasoning for critical judgment. Further research is required in this field or more realizations. However, this research is complete within its preset scope and can be used as a guideline for further research.

Bibliography

Alberts, B. (1989). Molecular biology of the cell. New York, Garland Pub.
Harris, Neil S, and William E. Winter. Multiple Myeloma and Related Serum Protein Disorders: An Electrophoretic Guide. New York, NY: Demos Medical Pub, 2012. Print.
Roswell, L. B. (2008). Protein conformation: new research. New York, Nova Science Publishers.
Shakhnovich, E. I. (2001). Protein folding, evolution and design: [proceedings of the NATO Advanced Study Institute on Protein Folding, Evolution and Design, 11-21 July, 2000, International School of Physics Enrico Fermi, Varenna (Como Lake), Italy]. Amsterdam [u.a.], IOS Press [u.a.].
Uversky, V. N., & Permi︠A︡Kov, E. A. (2007). Methods in protein structure and stability analysis. New York, Nova Biomedical Books.
Walker, J. M. (2002). The protein protocols handbook. Totowa, N.J., Humana Press. http://search.ebscohost.com/login.aspx?direct=true&scope=site&db=nlebk&db=nlabk&AN=67685.

Cite this page
Choose cite format:
  • APA
  • MLA
  • Harvard
  • Vancouver
  • Chicago
  • ASA
  • IEEE
  • AMA
WePapers. (2020, November, 01) Example Of Report On Practical 4. Retrieved August 03, 2021, from https://www.wepapers.com/samples/example-of-report-on-practical-4/
"Example Of Report On Practical 4." WePapers, 01 Nov. 2020, https://www.wepapers.com/samples/example-of-report-on-practical-4/. Accessed 03 August 2021.
WePapers. 2020. Example Of Report On Practical 4., viewed August 03 2021, <https://www.wepapers.com/samples/example-of-report-on-practical-4/>
WePapers. Example Of Report On Practical 4. [Internet]. November 2020. [Accessed August 03, 2021]. Available from: https://www.wepapers.com/samples/example-of-report-on-practical-4/
"Example Of Report On Practical 4." WePapers, Nov 01, 2020. Accessed August 03, 2021. https://www.wepapers.com/samples/example-of-report-on-practical-4/
WePapers. 2020. "Example Of Report On Practical 4." Free Essay Examples - WePapers.com. Retrieved August 03, 2021. (https://www.wepapers.com/samples/example-of-report-on-practical-4/).
"Example Of Report On Practical 4," Free Essay Examples - WePapers.com, 01-Nov-2020. [Online]. Available: https://www.wepapers.com/samples/example-of-report-on-practical-4/. [Accessed: 03-Aug-2021].
Example Of Report On Practical 4. Free Essay Examples - WePapers.com. https://www.wepapers.com/samples/example-of-report-on-practical-4/. Published Nov 01, 2020. Accessed August 03, 2021.
Copy

Share with friends using:

Please remember that this paper is open-access and other students can use it too.

If you need an original paper created exclusively for you, hire one of our brilliant writers!

GET UNIQUE PAPER
Related Premium Essays
Contact us
Chat now